Prokaryotic Recombinant Expression And Tissue-specific Expression Of Interleukins-10 And Interleukin-16 Genes In Orange-spotted Grouper(Epinephelus Coioides)

Interleukin-10(IL-10) and interleukin-16(IL-16) are pleiotropic cytokines and play important roles in specific immune and nonspecific immune. Recent studies indicated that there are IL-10 and IL-16 in fishes, and they play important roles in fish immunity..a orange-spotted grouper(Epinephelus coioides), an important marine fish in China, was studied in this paper. The full-length cDNA of the IL-10 gene in the grouper was cloned by using homological cloning and rapid ampli?cation of cDNA ends(RACE) methods. The results showed the IL-10 gene was 1104 bp long, with the open reading frame of 564 bp,encoding 187 amino acids. The estimated molecular weight and isoelectric point of the IL-10 protein were respectively 21.7 kDa and 5.74. The IL-10 was predicted with a signal peptide and without the transmembranous region. Homological analysis of the IL-10 showed that the grouper shared the high homology with IL-10 of Oreochromis niloticus.According to the known sequences of the interleukin-16(IL-16) genes in GenBank,homological cloning and rapid amplification of cDNA ends(RACE) methods, the full-length cDNA of the IL-16 gene in the grouper was cloned. The results showed the length of the IL-16 gene was 2859 bp, with the open reading frame of 1896 bp, encoding631 amino acids. The estimated molecular weight and isoelectric point of the IL-16 protein were respectively 68.2 kDa and 5.36. The IL-16 protein was with the signal peptide and without the transmembranous region. Based on the IL-16 protein, the homological analysis showed that the grouper shared the high homology with IL-10 of Larimichthys crocea.The IL-10 gene and the IL-16 gene were respectively connected with the pET-32a(+)vector to construct the expression plasmids pET32-IL10 and pET32-IL16. The recombinant fusive proteins IL-10 and IL-16 were induced to express in E. coli BL21(DE3) by isopropyl-β-thiogalactopyranoside(IPTG). SDS-PAGE showed that the aimed proteins were successfully expressed. The optimal conditions of the fusion proteins IL-10 and IL-16 expression were separately 37℃ for 3 hours, with 0.02 mmol/L IPTG and 37℃for 7 hours, with 0.2 mmol/L IPTG.In this study, the differential expressions of the IL-10 gene and the IL-16 gene in gill,thymus, head kidney, kidney, intestine, liver, spleen and brain of healthy fish and immunized fish with inactivated Vibrio hareyi were analyzed by fluorescent quantitativereal-time PCR technology(qRT-PCR) using β-actin gene as the control genes. qRT-PCR results showed that the IL-10 gene was mainly expressed in gill in healthy grouper After vaccination with inactivated V. hareyi 3 hr, the mRNA level of the IL-10 gene was up-regulated in head kidney, kidney, intestine, liver, spleen and brain. A clear time-dependent expression pattern of the IL-10 gene was showed after immunization.Expression of the IL-10 gene reached the highest level in head kidney and kidney on 6h post-vaccination, 12 h in gill and spleen. The IL-16 gene was expressed in all the studied tissues( gill, thymus, head kidney, kidney, intestine, liver, spleen and brain) of healthy grouper. The mRNA level of the IL-16 gene was down-regulated in the other studied tissues except thymus on 3 h post-vaccination with inactivated V. hareyi. There was no clear time-dependent expression pattern of the IL-16 gene after immunization. The expression of the IL-16 gene reached the highest level in spleen 12 h after vaccination.